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CN-114401844-B - Alkaline purification of spider silk proteins

CN114401844BCN 114401844 BCN114401844 BCN 114401844BCN-114401844-B

Abstract

The present disclosure relates to methods of producing and purifying synthetic block copolymer proteins, expression constructs for secreting the synthetic block copolymer proteins, recombinant microorganisms for producing the synthetic block copolymer proteins, and synthetic fibers comprising these proteins that reproduce many of the characteristics of natural filaments.

Inventors

  • Mei Jiaheng
  • R. B. mutalik
  • S.LI
  • S .zhan

Assignees

  • 保尔特纺织品公司

Dates

Publication Date
20260505
Application Date
20191126
Priority Date
20181128

Claims (20)

  1. 1. A method of isolating recombinant spider silk proteins from a host cell culture, comprising: a. obtaining a cell culture, wherein the cell culture comprises a host cell and a growth medium, wherein the host cell expresses a recombinant spider silk protein, wherein the recombinant spider silk protein comprises large ampullate gland 2 (MaSp 2) filaments, and wherein the cell culture comprises a fungal cell or a bacterial cell; b. Collecting a portion of the cell culture comprising the host cell and the recombinant spidroin protein without isolating the host cell from the recombinant spidroin protein; c. Incubating said portion of said cell culture in an aqueous solution under alkaline conditions having a pH in the range of 11-12 for a period of 15-60 minutes, thereby solubilizing said recombinant spider silk protein in said aqueous solution, and D. Separating the recombinant spidroin protein from the aqueous solution, thereby producing an isolated recombinant spidroin protein sample.
  2. 2. The method of claim 1, wherein the isolated recombinant spidroin protein is a full length recombinant spidroin protein.
  3. 3. The method of claim 2, wherein the isolated recombinant spidroin sample comprises at least 30% of the full length recombinant spidroin relative to the total isolated recombinant spidroin.
  4. 4. The method of claim 3, wherein the percentage of full length recombinant spider silk protein is measured using western blotting.
  5. 5. The method of claim 3, wherein the percentage of full length recombinant spider silk protein is measured using size exclusion chromatography.
  6. 6. The method of any one of claims 1-3, wherein the purity of the isolated recombinant spider silk protein is at least 30%.
  7. 7. The method of any one of claims 1-3, wherein the yield of the isolated recombinant spider silk protein ranges from 50-100% relative to recombinant spider silk isolated by urea or guanidine thiocyanate.
  8. 8. The method of any one of claims 1-3, wherein isolating the recombinant spidroin protein comprises precipitating the recombinant spidroin protein by altering the alkaline conditions of the aqueous solution.
  9. 9. The method of claim 8, wherein altering the alkaline condition comprises adjusting the alkaline pH of the portion of the cell culture to a reduced pH of from 4 to 10.
  10. 10. The method of claim 9, wherein the reduced pH is a pH of 4, 5, 6, 7, 8, 9, or 10.
  11. 11. The method of claim 9, wherein the reduced pH is a pH of from 6 to 7.
  12. 12. The method of claim 8, wherein adjusting the alkaline pH comprises adding an acid to the aqueous solution.
  13. 13. The method of claim 12, wherein the acid is H 2 SO4.
  14. 14. The method of any one of claims 1-3, wherein the portion of the cell culture comprises a supernatant, whole cell broth, or cell pellet.
  15. 15. The method of any one of claims 1-3, wherein collecting the portion of the cell culture comprises removing the host cells from the growth medium and reconstituting the host cells in the aqueous solution.
  16. 16. The method of any one of claims 1-3, wherein collecting the portion of the cell culture comprises lysing the host cells.
  17. 17. The method of claim 16, wherein lysing comprises heat treatment, shear disruption, physical homogenization, ultrasound, or chemical homogenization.
  18. 18. The method of any one of claims 1-3, wherein the portion of the cell culture comprises the host cell from the cell culture and the growth medium.
  19. 19. A method according to any one of claims 1 to 3, wherein the aqueous solution comprises a diluted growth medium.
  20. 20. The method of any one of claims 1-3, wherein incubating the portion of the cell culture under alkaline conditions is performed for 15 to 30 minutes.

Description

Alkaline purification of spider silk proteins RELATED APPLICATIONS The present application claims priority from U.S. provisional patent application No. 62/772,588 filed on 11/28 of 2018, the contents of which are incorporated herein by reference in their entirety. Sequence listing The present application comprises a sequence listing, which has been submitted through EFS-Web and incorporated herein by reference in its entirety. The ASCII copy was created on 10 months of 2019, named BTT-013WO_SL.txt, with a file size of 66,352 bytes. Background The silk polypeptides of spiders are large (> 150kDa, >1000 amino acids) polypeptides that can be broken down into three domains, an N-terminal non-repetitive domain (NTD), a repetitive domain (REP) and a C-terminal non-repetitive domain (CTD). NTD and CTD are relatively small (about 150, about 100 amino acids, respectively), well studied, and are believed to confer water stability (aqueous stability), pH sensitivity, and molecular alignment after aggregation to the polypeptide. NTD also has a strong predictive secretion tag that tends to be removed during heterologous expression. The repeat regions account for about 90% of the native polypeptide and fold into crystalline and amorphous regions that impart strength and flexibility, respectively, to the silk fiber. Silk polypeptides come from a variety of sources, including bees, moths, spiders, mites, and other arthropods. Some organisms produce a variety of silk fibers with specific sequences, structural elements, and mechanical properties. For example, a circular mesh (orb weaving) spider has six unique types of glands that produce different silk polypeptide sequences that can be polymerized into fibers suitable for the environment or life cycle microenvironment (niche). The fiber is named by the gland from which it originates, and the polypeptide is labeled by the gland abbreviation (e.g. "Ma") and the "Sp" of spider silk protein (short for spider silk fibroin). In the circular spider, these types include the large ampullate gland (MaSp, also known as dragline), the small ampullate gland (MiSp), the whip gland (Flag), the grape gland (AcSp), the tubular gland (TuSp), and the piriform gland (PySp). This combination of polypeptide sequences that span fiber types, domains, and variations between different organisms and species creates a variety of potential properties that can be exploited by commercial production of recombinant fibers. To date, most of the work on recombinant silk has focused on major ampullate spidroin proteins (MaSp). Currently, recombinant silk fibers are not commercially available (with few exceptions) and cannot be produced in microorganisms other than escherichia coli (ESCHERICHIA COLI) and other gram-negative prokaryotes. Recombinant filaments produced to date consist mainly of polymerized short filament sequence motifs or fragments of the original repeat domains, sometimes combined with NTD and/or CTD. This results in small scale production of recombinant silk polypeptides using intracellular expression (milligrams on a laboratory scale and kilograms on a bioprocessing scale) and purification by chromatography or bulk precipitation. These methods do not produce viable commercial scalability that can compete with the price of the prior art and textile fibers. Additional production hosts that have been used to produce silk polypeptides include transgenic goats, transgenic silkworms, and plants. These hosts have not been able to produce silk on a commercial scale, possibly due to slow engineering cycles and poor scalability. In addition, recombinant silk polypeptides form undesirable insoluble aggregates during production and purification. The process of re-solubilising the peptide during purification tends to degrade the protein, resulting in lower fiber yields, lower fiber toughness and poorer hand. Furthermore, standard protein solubilization methods require the use of chaotropic agents (chaotrope), such as urea, guanidine hydrochloride, or guanidine thiocyanate, which must be collected and properly disposed of after protein separation. Thus, there is a need for improved methods to purify these polypeptides in a sustainable and environmentally friendly process. Disclosure of Invention In one aspect, provided herein is a method of isolating recombinant spidroin from a host cell culture comprising obtaining a cell culture, wherein the cell culture comprises a host cell and a growth medium, wherein the host cell expresses the recombinant spidroin, collecting a portion of the cell culture comprising the recombinant spidroin, incubating the portion of the cell culture in an aqueous solution under alkaline conditions, thereby solubilizing the recombinant spidroin in the aqueous solution, and isolating the recombinant spidroin from the aqueous solution, thereby producing an isolated recombinant spidroin sample. In some embodiments, the alkaline conditions comprise an alkaline pH of 9 to 14. In one emb